1. Field of the Invention
The present invention relates to a fluorescent confocal microscope so designed that the surface of a specimen is scanned by an excitation light whereby the resulting fluorescent light emanating from the specimen is condensed on each of pinholes and the fluorescent light passed through the pinholes is detected, thereby selectively obtaining only focusing information.
2. Related Background Art
With a conventional fluorescent confocal microscope, a microscope objective lens compensated for changes in the chromatic aberration of magnification for purpose of the ordinary observation with the naked eye or a television camera is employed as its objective lens.
In the past, while, in the case of the system of a microscope objective lens unit, the composite system of a microscope objective lens and an eyepiece according to the compensation method and the like, a chromatic aberration of magnification compensation has been provided for with respect to two particular wavelengths, e.g., the F line (blue) and the C line (red), there have existed the secondary spectra of chromatic aberrations of magnification with respect to the other wavelengths.
Then, in the case of such conventional fluorescent confocal microscope, as in the case in many instances, if the wavelengths of the excitation light and the fluorescent light fail to be coincident with the particular wavelengths subject to the chromatic aberrations of magnification compensation of the objective lens, chromatic aberrations of magnification appear at the excitation light wavelength and the fluorescent light wavelength.
In the case of the ordinary fluorescent microscope, such chromatic aberration of magnification do not present much problems since they merely change the magnification of a fluorescent image. In the case of the fluorescent confocal microscope, however, if there is the difference in chromatic aberration of magnification between the excitation light wavelength and the fluorescent light wavelength, the center of the fluorescent light spot deviates from the center of the pinhole so that the light quantity passed through the pinhole is decreased and the image becomes darker.
Since the chromatic aberration of magnification becomes increasingly large from the center of the visual field to the adjacent regions on the whole, even if the object (specimen) emits the same amount of fluorescent light in both the center and adjacent regions of the visual field, the image produced by the fluorescent confocal microscope becomes such that the image is darker in the adjacent regions than in the center of the visual field.
Particularly, there is a problem that if the diameter of the pinhole is decreased to obtain an optical cutting function and an excellent lateral resolution which are features of the fluorescent confocal microscope, the light quantity is decreased extremely in the adjacent regions of the visual field and only the center and its vicinity of the visual field are practically observed, thereby narrowing the visual field.
Moreover, if the center of the fluorescent light spot deviates from the center of the pinhole, the form of the detected light quantity distribution relative to the displacement of the specimen in the direction of the optical axis is changed and the reliability of the resulting image is deteriorated.
Further, where a fluorescent reagent (Indo-1) of a single excitation light wavelength and two fluorescent light wavelengths, a fluorescent reagent (Fura-2) of two excitation light wavelengths and a single fluorescent light wavelength or the like is used to make a quantitative measurement of ions or the like by a comparison in light quantity between the excitation light of the single wavelength and the fluorescent light of the two different wavelengths or a comparison in light quantity between the excitation light of the two different wavelengths and the fluorescent light of the single wavelength, there is a problem that if there is the difference in the magnitude of chromatic aberration of magnification between the two excitation wavelengths or the two fluorescent light wavelengths, the difference in the degree of decrease in light quantity between the two different excitation light wavelengths or fluorescent light wavelengths increases from the center to the adjacent regions of the visual field, thereby ruining the quantitativeness of the measurement.